Naturally occurring proteins acquire increased activity through the repeated evolutionary cycle of selection, mutation and amplification of genes. In the organism, most proteins are modified for variation of their functional groups and therefore exhibit considerable functional variation. The advent of evolutionary molecular engineering of proteins has led to artificial creation of proteins or of genetic DNA encoding therefor that form the basis of bioreactions, in the laboratory, for industrial use. The technology has made possible the emergence of enzymes and proteins exhibiting new activity not found in nature, or proteins with significantly different structures from natural proteins, which are expected to have a variety of applications in the fields of medicine and engineering. Evolutionary molecular engineering involves the selection of molecules having desired activity from among a random polymer pool of block units of amino acids or nucleotides making up proteins or the genes encoding them.
However, it has been desired to increase protein functional group variation, and to develop additional protein immobilization and protein stabilization techniques suitable for bioprocessing, in order to achieve further advances in the field. The key to such advances is protein modification technology. For example, modification of a protein used in treatments for hepatitis, interferon, with polyethylene glycol (PEG) has successfully increased in vivo stability and lengthened elimination half-life in blood, even when the modification is nonspecific. Modification techniques are also very similar to protein immobilization techniques, that are important for bioprocessing and for increased functionality of cell culture dishes. In other words, the difference between the techniques is simply whether the substance added to the protein by modification is smaller than a given size, or whether it is a support.
Methods for site-specific modification of proteins that have become known in recent years include a method of designing a mutant having all of the lysine residues of a protein replaced or a mutant having all but one of the cysteine residues replaced, in order to limit the modification site to a single location (for example, see Nature Biotechnology, 2003, vol. 21, pp. 546-552). However, large-scale amino acid-substitution that replaces a given type(s) of amino acid at most of all sites, has been associated with the drawback of reduced protein activity. Also, major effort has been required to explore mutants that compensate for the reduced activity.
In a reported system for development of the method described above, a phage display is used to select a protein that has no lysine residues but still retains activity, from among an initial library wherein all the lysine codons at six locations are randomized. However, because only a very few clones had all of the lysines replaced, it is expected that the efficiency of obtaining active clones with lysines replaced will be even lower for other proteins in general. Also, it is assumed that unintended mutations during the course of preparing the library resulted in lysine codons even at sites that did not code for lysine in the original protein. In fact, the activities of the clones obtained in the aforementioned report were only moderate.
There are also known methods wherein a protein synthesis system containing an added aaRS (aminoacyl tRNA synthase) mutant is used to introduce amino acids other than the usual 20 (non-naturally occurring amino acids) during protein synthesis (for example, see Japanese Unexamined Patent Publication (Kokai) No. 2004-261160, International Patent Publication No. WO 03/014354, and PNAS Jan. 7, 2003, vol. 100, No. 1, pp. 56-61). In the former cited document, production of a protein with introduction of highly reactive non-naturally occurring amino acids containing ketone groups or the like is followed by reaction of the functional groups for modification (PNAS Jan. 7, 2003, vol. 100, No. 1, pp. 56-61). In the latter cited documents, non-naturally occurring amino acids with the intended modifications are introduced into the protein directly on the ribosome (Japanese Unexamined Patent Publication (Kokai) No. 2004-261160, and International Patent Publication No. WO 03/014354). These methods have the advantage of allowing site-specific modification without reduction in activity, and without the use of evolutionary molecular engineering techniques.
However, since it is rarely possible to obtain the desired properties simply by site-specific mutagenesis using rational design of protein mutants, and most proteins exhibit reduced activity as a result, evolutionary molecular engineering must be applied for practical results. For mass production, such methods must employ protein synthesis systems with yet additional special features. Other major problems are that the fidelity of such protein synthesis systems is low, and the proteins obtained as industrial products are not homogeneous. One of the methods also requires preparation of a specialized aaRS mutant for each type of non-naturally occurring amino acid. Preparation of such mutants is not only difficult currently, but the non-naturally occurring amino acids are also limited in their physical size in order to be acceptable for the various aspects of protein synthesis.